Validation of a rapid collagenase activity detection technique based on fluorescent quenched gelatin with synovial fluid samples

Background Measuring collagenase activity is crucial in the field of joint health and disease management. Collagenases, enzymes responsible for collagen degradation, play a vital role in maintaining the balance between collagen synthesis and breakdown in joints. Dysregulation of collagenase activity leads to joint tissue degradation and diseases such as rheumatoid arthritis and osteoarthritis. The development of methods to measure collagenase activity is essential for diagnosis, disease severity assessment, treatment monitoring, and identification of therapeutic targets. Results This study aimed to validate a rapid collagenase activity detection technique using synovial fluid samples. Antibody microarray analysis was initially performed to quantify the levels of matrix metalloproteinase-9 (MMP-9), a major collagenase in joints. Subsequently, the developed gelatin-based test utilizing fluorescence measurement was used to determine collagenase activity. There was a significant correlation between the presence of MMP-9 and collagenase activity. In addition, Lower Limit of Detection and Upper Limit of Detection can be preliminary estimated as 8 ng/mL and 48 ng/mL respectively. Conclusions The developed technique offers a potential point-of-care assessment of collagenase activity, providing real-time information for clinicians and researchers. By accurately quantifying collagenase activity, healthcare professionals can optimize patient care, improve treatment outcomes, and contribute to the understanding and management of joint-related disorders. Further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.


Background
Measuring collagenase activity is of utmost importance in the field of joint health and disease management.Collagenases, a group of enzymes responsible for the degradation of collagen, play a critical role in maintaining the balance between collagen synthesis and breakdown within connective tissues within the joint [1,2].If the homeostasis controlled by collagenases is disrupted, joint tissue degradation is provoked, causing joint diseases such as rheumatoid arthritis (RA) or osteoarthritis (OA) [3][4][5].This imbalance results in the progressive erosion of articular cartilage and structural alterations.
Collagenases belong to the matrix metalloproteinase (MMP) family of proteins.The MMP family consists of various enzymes that are involved in the degradation and remodeling of the extracellular matrix (ECM), including collagen.Collagenases, including MMP-1, MMP-8, and MMP-13, primarily cleave collagen fibrils.Gelatinases, represented by MMP-2 and MMP-9, target denatured collagen (gelatin) as well as other ECM constituents.Stromelysins, such as MMP-3, MMP-10, and MMP-11, are involved in the cleavage of various ECM proteins, including proteoglycans and fibronectin.Membrane-type MMPs, exemplified by MT1-MMP, participate in the activation of proMMP-2 and proMMP-13.This orchestrated enzymatic activity is crucial for tissue development, repair, and homeostasis.However, dysregulation of MMPs is associated with pathological conditions such as cancer, arthritis, and cardiovascular diseases.
Therefore, understanding the specific targets of each MMP is essential for therapeutic strategies aimed at modulating ECM remodeling and preventing aberrant tissue changes.Among them, matrix metalloproteinase 9 (MMP-9), also known as gelatinase B, plays a significant role in various physiological and pathological processes in joints.MMP-9 is primarily involved in the breakdown of fibrillar collagens, particularly type IV and V collagens, which are components of the ECM in joint tissues.It cleaves collagen molecules at specific sites, resulting in the fragmentation and degradation of collagen fibers.During inflammatory processes, such as those of RA or OA, MMP-9 is upregulated as part of the innate immune response.It not only contributes to collagen degradation but also promotes the migration of immune cells to the site of inflammation and facilitates the release of cytokines and chemokines, amplifying the inflammatory cascade [6,7].
In general, collagenases, including matrix metalloproteinase-1 (MMP-1), MMP-8, MMP-9, and MMP-13, play a crucial role in the molecular basis of both RA and OA [8].These collagenases contribute to the degradation of collagen within the joint, particularly collagen type II, which is a major component of articular cartilage.Increased levels and activity of MMP-1, MMP-8, MMP-9, and MMP-13 in SF are associated with ongoing joint tissue degradation and can serve as diagnostic biomarkers for both RA and OA [9,10].
In this context, collagenases have emerged as potential therapeutic targets for the treatment of joint diseases.Modulating collagenase activity can help regulate collagen degradation, maintaining the balance between collagen turnover and tissue preservation.Researchers are exploring strategies to selectively inhibit or modulate collagenase activity, aiming to slow joint tissue degradation and potentially delay the progression of joint diseases [9,10].
In particular, MMPs play a significant role in the homeostasis of synovial fluid (SF), particularly in the context of joint health and disease [20].In the synovial joints, MMPs are involved in the dynamic turnover and remodeling of the ECM within the SF, a specialized lubricating fluid that surrounds and nourishes the joints [20].These enzymes contribute to the degradation of collagen, gelatin, and proteoglycans within the SF, influencing the viscosity and composition of this fluid.They are produced by synovial cells and chondrocytes, degrade various ECM molecules (Table 1) maintaining the fluidity and functionality of the synovial fluid, ensuring proper joint lubrication and shock absorption [21][22][23][24][25]. MMPs are also influenced by Calcium, Ca2 + ions are crucial for the catalytic domains of MMPs, playing a vital role in the proper folding of the molecule necessary for substrate recognition and catalysis [26].Furthermore, this element influences the activity of MMPs through various mechanisms, including binding to calmodulin, modulating integrin-mediated signaling, and participating in intracellular cascades like PKC activation [27].It also impacts MMP gene expression by regulating transcription factors such as NFAT and AP-1 [28].In addition, MMPs are tightly regulated by tissue inhibitors of metalloproteinases (TIMPs), balancing ECM synthesis and degradation.This balance facilitates the removal of damaged matrix components and supports the synthesis of new ones, thereby contributing to joint health and the prevention of diseases such as osteoarthritis [29,30].Under normal physiological conditions, MMPs help maintain the delicate balance between ECM synthesis and degradation, ensuring proper joint function.However, in conditions such as RA and OA, there is often an imbalance in MMP activity, leading to excessive degradation of the ECM in the SF.This dysregulation contributes to joint damage, inflammation, and the progression of joint diseases.Understanding the intricate interplay between MMPs and SF dynamics is crucial [30,31].Therefore, measuring collagenase activity plays a crucial role in diagnosing joint diseases, assessing disease severity, monitoring treatment response, and identifying therapeutic targets [32][33][34].These measurements provide valuable information for clinicians, researchers, and drug developers, ultimately contributing to the understanding and management of joint-related disorders [32][33][34].By accurately quantifying collagenase activity, health care professionals can optimize patient care, improve treatment outcomes, and strive to preserve joint health and functionality.The development of a rapid test to assess collagenase activity in SF holds significant importance in the field of joint health and disease management.Currently, the measurement of collagenase activity often involves complex laboratory techniques that require specialized equipment and time-consuming procedures.Among these, widely used tests include zymography, which visually assesses enzyme activity through gel electrophoresis, and fluorogenic assays, which quantitatively measure collagenase activity using specific substrates [35,36].Among these substrates, DQ-gelatin, FRET-based peptides, or fluorogenic peptides derived from collagen sequences, such as those containing MCA (7-methoxycoumarin-4-acetyl) and DNP (dinitrophenyl) groups, have been widely used in the study of collagenase activity as they release a fluorescent signal upon cleavage by the enzyme, allowing for real-time and quantitative measurement of enzyme activity [37][38][39][40][41][42].Immunoassays, such as ELISAs, which detect collagenase antigens, also play a crucial role in providing quantitative insights [43].These diagnostic tools are invaluable in diagnosing and monitoring conditions associated with collagen degradation, aiding clinicians in making informed decisions about patient management.However, a rapid test could revolutionize the diagnostic process by providing real-time, point-of-care (PoC) assessment of collagenase activity, offering numerous advantages and potential applications.
This work aimed to validate a developed rapid collagenase activity detection technique.This test is based on immobilizing fluorescent quenched gelatin (Gel-FITC) at the bottom of the wells of a microplate.When incubated with synovial fluid, the collagenases in the fluid digest the gelatin, liberating the fluorophore and increasing the fluorescent signal, which, when measured together with a calibration curve, allows the quantification of the collagenase activity of the sample normalized by the presence of the enzyme.We validated this test by comparing the collagenase activity of 22 samples with the presence of MMP-9 previously calculated with antibody microarrays (AbMAs).
The novelty of this research lies in the innovative utilization of fluorescent quenched gelatin as a substrate for collagenase detection.By immobilizing this gelatin at the bottom of microplate wells, a platform that allows for efficient and rapid assessment of collagenase activity has been created.The principle behind this technique, wherein collagenases present in synovial fluid digest the gelatin substrate, liberating a fluorophore and resulting in an increase in fluorescent signal, showcases a useful integration of molecular biology and spectroscopic methods, which offers to researchers and clinicians a valuable tool for studying enzyme kinetics and diagnosing conditions associated with collagen degradation.
The novel Gel-FITC degradation assay, which takes about 30 min, is significantly faster and requires fewer resources compared to traditional methods like zymography, fluorogenic assays, and ELISAs, which take several hours to a full day.This makes the Gel-FITC test an efficient and accessible option for real-time, point-of-care collagenase activity assessment.We selected Gel-FITC for its accessibility, ease of manufacturing, affordability, and stability, making it a practical choice for widespread use in real-time collagenase activity assessment, especially in resource-limited settings.Its simplicity and low cost align with the need for efficient diagnostic tools that can be readily deployed in various healthcare settings.
While the utilization of fluorescent quenched gelatin for collagenase detection presents promising advantages, it is also crucial to acknowledge its potential drawbacks and limitations.It should be noted that the technique's sensitivity may vary depending on several factors, including the concentration of collagenases in the sample and the efficiency of the gelatin substrate immobilization.In addition, there is a potential batch-to-batch variation in the gelatin substrate, which might affect assay performance and result interpretation.In order to solve all this disadvantages, when verifying, validating and upscaling the manufacturing, effective quality control measures and standardized protocols would be necessary to ensure assay consistency.Furthermore, the need for specialized equipment, such as microplate readers capable of detecting fluorescence, could limit the accessibility of the technique, particularly in resource-limited settings or smaller research laboratories.Despite these drawbacks, the innovative nature of the technique and its potential applications in enzyme kinetics and disease diagnosis warrant further investigation and optimization to maximize its utility and address existing limitations.Continued refinement and validation efforts will be essential to enhance the reliability and robustness of this collagenase detection method.

Synovial fluid samples
We designed a validation study in which 22 samples were analyzed.SF samples were obtained from patients who presented with joint effusion due to degenerative knee pathologies.This effusion is extracted in routine medical practice prior to the administration of intra-articular treatments.The samples were aliquoted in 0.5 mL Eppendorf tubes (#40420050, Hamburg, Germany) and stored at − 80 °C until analysis.The collection and processing of samples was carried out by medically qualified personnel after approval from the institutional review board and in strict accordance with the tenets of the Helsinki Declaration regarding research in humans.The corresponding ethical approval was obtained (Protocol No. EPA2015046) from the Ethics Committee of the Basque Country (September 2015).Informed consent was obtained from all the participants after the nature and possible consequences of the study were explained to them.
The immunodetection protocol involved first thawing and drying the slides for 30 min at RT in a drying chamber, after which they were then washed three times for 5 min with phosphate-buffered saline containing 0.01% Tween-20 (0.01% PBS-T) with agitation.The AbMAs were left in blocking solution (2.5% milk powder in 0.01% PBS-T) for 10 min at RT and then washed with distilled water.The AbMAs were then incubated overnight at 4 °C in a humid chamber with the samples diluted 1:10 in 0.5% PBS-T and 0.01% sodium dodecyl sulfate (SDS: #436143, Sigma, St. Louis, MA, USA).Alternatively, the slides were probed with the biomarker standard (MMP-9, #10327-HNAH, Sino Biological, Beijing, China) at the desired concentrations to establish calibration curves (Fig. 2).
A final volume of 20 μL was used for each AbMA.After probing, the slides were washed twice with 0.5% PBS-T and once with 0.01% PBS-T for 10 min each with agitation.The AbMAs were probed for 1 h at RT in a humid chamber with primary rabbit IgG antisera against human MMP-9 (10 μg/mL, diluted in blocking solution).After incubation, the slides were washed once with 0.5% PBS-T and twice with 0.01% PBS-T for 5 min each with agitation.The slides were incubated for 1 h at RT in a humid chamber with an Alexa Fluor 647-conjugated secondary goat anti-rabbit IgG antibody (#ab150079: Abcam, Cambridge, UK) diluted 5 μg/mL in blocking solution.Subsequently, the slides were washed with agitation for 5 min

Collagenase activity of the samples
To develop a method to measure collagenase activity in human samples, gelatin-fluorescein (Gel-FITC) conjugate (#M1303, BioVision, Milpitas, CA, USA) was chosen as a substrate for the detection of gelatinases/collagenases and other gelatin-degrading enzymes.This gelatin contains the fluorophore FITC, which is quenched until the gelatin is digested when the fluorophore is released.We used P386 well microplates from Greiner (#781289, Greiner, Madrid, Spain) for the immobilization of the gelatin following the protocol detailed below.
First, Gel-FITC was dissolved in 200 μL of distilled water, resulting in a 5 mg/mL solution, which was kept in a rotary mixer for 30 min.Then, the solution was frozen at -20 °C in 25 aliquots of 5 μL.Before use, the 5 mg/ mL Gel-FITC solution was thawed at RT and diluted to a final concentration of 19 μg/mL in 0.1 M PB, pH 7.6.The solution was mixed in a rotary mixer protected from light for 30 min.Next, 50 μL of the solution was pipetted into each of the wells of the P386 microplate, and the plate was dried overnight in an oven at 40 °C for the immobilization of the Gel-FITC.Before incubation, 100 μL of enzyme incubation buffer (50 mM Tris HCl, 150 mM NaCl, 5 mM CaCl2, and 0.01% Tween, pH 7.6) was pipetted into each well of the plate for washing, the plate was incubated for 5 min in an orbital shaker, and the plate was inverted to empty the wells until the incubation buffer was removed.Finally, the samples were incubated 1:1 in enzyme incubation buffer at a final volume of 50 μL per well.Alternatively, the wells were probed with the collagenase standard (MMP-9, #10327-HNAH, Sino Biological, Beijing, China) at the desired concentrations in incubation buffer to establish calibration curves (Fig. 2).The fluorescent signal was read at 485/540 nm at 37 °C for 20 min with lectures every 2 min using a Thermo Scientific Fluoroskan Ascent FL (#1506450, Thermo Fisher Scientific, Waltham, MA, USA).A schematic representation of how this test was performed is presented in Fig. 3.

Statistical analysis
Simple bivariate correlation was calculated when comparing Gel-FITC digestion measurements and AbMA quantifications, setting the significance at α = 0.05 using a two-tailed test for validating the reliability of the test.The data were processed with GraphPad Prism 9.2.0 (Graph-Pad Software, San Diego, CA, USA).Statistical analyses were carried out with SPSS 23.0 software (IBM, Armonk, NY, USA).

Analysis of the MPP-9 concentration in synovial fluid samples by antibody microarrays
The biomarker MMP-9 was analyzed in 22 synovial liquid samples using AbMAs.The concentration of each biomarker was assessed (Fig. 4), and the mean, median, maximum and minimum values were calculated (Table 2).The MMP-9 concentration was determined with AbMAs according to specific protocols.The mean concentration of MMP-9 calculated was 12.42 ng/mL, with a standard error (SEM) of 1.08 ng/mL.The median value was 11.08 ng/mL.The maximum concentration observed in the dataset was 21.89 ng/mL, and the minimum concentration was 3.78 ng/mL.

Determination of collagenase activity in synovial fluid samples
The collagenase activity of 22 synovial liquid samples was calculated by Gel-FITC digestion.The activity of collagenases normalized by enzyme presence was assessed (Fig. 5), and the mean, median, maximum and minimum values were calculated (Table 3).Collagenase presence was calculated by Gel-FITC digestion, obtaining a mean value of 26.45 ng/mL, with a standard SEM of 3.26 ng/ mL.The median value was 26.85 ng/mL.The maximum concentration observed in the dataset was 48.75 ng/ mL, and the minimum concentration was 0.00 ng/mL.In addition, Lower Limit of Detection (LLoD) can be preliminarily estimated as 8 ng/mL as is the lowest collagenase concentration detected when analyzing synovial liquid clinical samples with the developed activity assay.In the same way, Upper Limit of Detection can be preliminarily estimated as 48 ng/mL (Fig. 5).

Comparison between MMP-9 concentration and collagenase activity
To assess the reliability of this technique, the normalized collagenase activity values obtained were compared with the concentrations of MMP-9 collagenase determined by AbMAs in the same samples.When comparing both values, similar results were obtained (Fig. 6).Additionally, a simple bivariate correlation was calculated, setting the significance at α = 0.05 using a two-tailed test.A Pearson correlation coefficient (r) of 0.5459 was obtained with a significance of (*), and the p value was 0.0156 (p value < 0.05).

Discussion
We developed a rapid test for immobilizing Gel-FITC at the bottom of the wells of a microplate and established a detection protocol for the quantification of collagenase activity in SF.The purpose of this test is the rapid quantification of collagenase activity as an instrument for evaluating joint diseases.
As introduced in the Background section, Gel-FITC is highly suitable due to its ease of accessibility, manufacturability, affordability, and stability.It has been used successfully in different works [50,51].Other substrates that can be used instead include fluorescently labeled   peptides or proteins [37][38][39][40][41][42].However, these alternatives might not have been widely adopted due to factors such as higher costs, limited availability, or lesser stability compared to Gel-FITC.Additionally, Gel-FITC's simplicity and effectiveness make it a preferred choice for many applications.
Our results validated the developed test by comparing the normalized collagenase activity calculated by Gel-FITC digestion with the MMP-9 concentration calculated by using AbMAs.For this purpose, 22 synovial liquid samples were collected and processed using both techniques.After quantification, the observed ranges of enzyme expression and collagenase activity obtained with AbMAs and Gel-FITC, respectively, were within the ranges described in the literature, which are between 0 and 30 ng/mL [20,50,51].It should be noted that the normalized collagenase activity values were greater than the MMP-9 values quantified by AbMAs.This is because not only MMP-9 but also multiple MMPs have collagenolytic activity.Other specific collagenases within the MMP family are known as MMP-1, MMP-8, and MMP-13.These collagenases, which are present in SF, are characterized by their ability to cleave and degrade fibrillar collagens, particularly type I, II, and III collagens, which are abundant in connective tissues, including those found in joints [6].
To validate the Gel-FITC degradation test, a correlation between the collagenase activity calculated by Gel-FITC digestion and the presence of MMP-9 calculated with AbMAs was performed.A statistically significant correlation in the obtained values was observed, indicating that the developed test is a useful and reliable tool for quantifying collagenase activity in human synovial liquid samples.
Collagenase activity measurements can aid in the diagnosis of joint diseases, particularly those characterized by collagen degradation, such as RA and OA.Elevated collagenase activity in the SF may indicate an imbalance in collagen turnover and degradation, indicating ongoing tissue destruction and inflammation [52][53][54].Moreover, measuring collagenase activity in SF can also help differentiate between different types of arthritis.RA, for instance, is characterized by excessive synovial inflammation and upregulation of collagenase activity [55].On the other hand, OA, a degenerative joint disease, typically exhibits lower levels of collagenase activity [34].These distinctions can assist in accurately diagnosing and classifying specific arthritic conditions, leading to more targeted and effective therapeutic approaches.In addition, measuring collagenase activity provides insights into the extent and severity of collagen degradation within the joint [56].Quantifying collagenase activity levels can help gauge the magnitude of tissue destruction, allowing Fig. 6 Correlation between the concentration of collagenases (ng/mL) calculated by Gel-FITC digestion using the normalized collagenase activity using an MMP-9 reference and the concentration of MMP-9 calculated with AbMAs (ng/mL) healthcare professionals to assess disease severity and monitor disease progression over time.Higher levels of collagenase activity may indicate a more aggressive disease course and predict a poorer prognosis [57,58].This information can guide treatment decisions and provide prognostic indicators for patients, assisting in determining the appropriate course of action and potential interventions.Furthermore, by periodically measuring collagenase activity, healthcare providers can evaluate the effectiveness of treatments aimed at reducing joint inflammation and preventing collagen degradation.Tracking changes in collagenase activity over time provides valuable feedback on the impact of interventions, allowing for treatment adjustments and optimization of patient care [59].
With these types of collagenase activity tests, researchers can investigate the impact of various factors, such as inflammation or oxidative stress, on collagenase activity levels, providing insights into the underlying pathophysiology of joint diseases [60][61][62].Moreover, these findings -No yet validated with various types of samples.
-Real-time monitoring of enzyme activity.
-Difficult to provide information about the identity of the analyzed collagenases.
Enzyme-Linked Immunosorbent Assay (ELISA) [70,71] ELISA involves the immobilization of a substrate specific to collagenase on a solid surface, followed by detection using an enzyme-linked antibody.
-Well-established method with commercial availability.
-Time-consuming procedure requiring several hours to complete.-High specificity for collagenase detection.
-Limited dynamic range, challenging for samples with low enzyme activity.-Compatible with various sample types and formats.
-Reliance on antibodies and substrates may introduce batch-to-batch variability and increase assay costs.
-Quantitative results achievable with appropriate calibration standards.
-Sensitivity may vary depending on antibody affinity and substrate efficiency.Western Blotting [72,73] Western blotting involves separating proteins from a sample by gel electrophoresis, followed by transfer to a membrane and detection using antibodies specific to collagenase proteins.
-Established methodology with documented protocols.
-Requires specialized equipment and expertise for gel electrophoresis and membrane transfer.
-High specificity for detecting collagenase proteins.
-Time-consuming and labor-intensive procedure.
-Suitable for analyzing protein expression levels and post-translational modifications.
-Sensitivity may vary depending on antibody quality and detection method.Zymography [35,74,75] Zymography involves embedding a substrate specific to collagenase in a gel matrix, followed by separation by gel electrophoresis and incubation to allow enzyme activity, visualized as clear bands.
-Specifically designed for detecting enzyme activity.
-Gel-based technique may not provide quantitative data.
-Allows visualization of active enzyme bands directly on the gel.
-Sensitivity may vary depending on gel composition and staining method.
-Can detect multiple enzymes simultaneously.
-Limited resolution for distinguishing closely related enzymes.Fluorescence Resonance Energy Transfer (FRET) Assays [39][40][41][42] FRET assays utilize changes in fluorescence intensity between fluorophores linked by a peptide sequence susceptible to collagenase cleavage to detect enzymatic activity in real-time.
-Sensitive detection of enzymatic activity based on changes in fluorescence intensity.
-Design and optimization of FRET substrates may be challenging.
-Real-time monitoring of enzyme activity.
-Background fluorescence and assay conditions may affect signal-to-noise ratio.-Potential for high-throughput screening applications.
-Requires specialized equipment for fluorescence detection.Mass Spectrometry [76] Mass spectrometry is used to detect and quantify peptide fragments generated by enzymatic cleavage of substrates, providing information about the identity and quantity of collagenase activity in a sample.
-High sensitivity and specificity for detecting peptide fragments generated by enzymatic cleavage.
-Expensive instrumentation and expertise required.
-Can provide information about the identity and quantity of collagenase activity in a sample.
-Sample preparation may be complex and time-consuming.
-Suitable for identifying novel enzyme substrates.
-Interpretation of mass spectrometry data may require bioinformatics expertise.can guide the development of novel therapeutic interventions targeting collagenase or its regulatory mechanisms, potentially leading to the discovery of more effective treatments [63].
One of the strengths of the test developed is its speed and simplicity, which allows it to position itself as a valuable PoC.Detecting elevated collagenase activity levels in SF at an early stage may indicate ongoing joint tissue degradation before clinical symptoms become apparent.This early intervention can lead to more effective treatment strategies, helping to prevent further joint damage and improve long-term prognosis [64][65][66].
However, further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.To date, collagenase activity measurements should be interpreted in conjunction with other clinical parameters and imaging studies to form a comprehensive diagnostic picture.Combining collagenase activity analysis with imaging techniques such as magnetic resonance imaging (MRI) or ultrasound can provide a more complete assessment of joint health, aiding in accurate diagnosis and treatment planning [67][68][69].
With the aim of summarizing the main advantages of the developed Gel-FITC collagenase activity assay in comparison to current techniques with the same intended use, the following table (Table 4) has been constructed.
In summary, the development of a rapid test to assess collagenase activity in SF holds immense importance in joint health.It can enable early diagnosis, aid in treatment monitoring, facilitate personalized care, identify therapeutic targets, and advance research in the field.By providing quick and reliable results, a rapid collagenase activity test has the potential to revolutionize the diagnosis and management of joint diseases, leading to improved patient outcomes and enhanced quality of life.In order to market current Gel-FITC collagenase assay, full package of characterization studies will be performed following CLSI guidelines to determine: Linearity (EP06-A), Detection Capability (EP17-A2), Precision (EP05-A3), Interfering substances (EP07-A3), Method Comparison (EP09-A3/EP12-A2), Bias (EP10-A3), and Stability (EP25-A) [77].

Conclusion
A rapid Gel-FITC degradation test was developed for synovial liquid collagenase activity quantification in human samples.The test was validated through comparison with AbMAs.With the obtained data, we confirmed the reliability of our Gel-FITC test for the quantification of collagenase activity in human synovial liquid samples.The measurement of this parameter offers a valuable diagnostic tool for assessing joint health and detecting various arthritic conditions.By providing insights into collagen turnover and tissue destruction, these measurements can assist in early diagnosis, differentiation between different types of arthritis, and monitoring treatment response.As research continues to unveil the intricacies of collagenase activity, it holds immense potential to revolutionize the diagnosis and management of jointrelated disorders, leading to improved patient outcomes and enhanced quality of life.

Fig. 1
Fig. 1 Schematic representation of a glass slide with printed AbMAs.Twenty-four AbMAs with two spots of rabbit IgG anti-human MMP-9 at 200 μg/mL in 0.05% SIVG were immobilized onto treated slides.Image created with BioRender.com

Fig. 2
Fig. 2 Schematic diagram of standard curve generation.Serial dilutions are made starting from a stock aliquot of 500 ng/mL of MMP-9.The volumes marked over the arrow are taken from the previous dilution and pipetted onto the previously prepared volumes marked on the bottom of the eppendorf.The final MMP-9 concentrations are indicated at the bottom of the figure

Fig. 3 A
Fig.3A brief summary of the protocol for the detection of the collagenase activity of the samples using quenched Gel-FITC.(A) Gel-FITC was added to the bottom of the wells containing P386.The samples were then loaded into each of these wells, whereby collagenases such as MMP-9 digest the gelatin, releasing the fluorophore and increasing the fluorescent signal.(B) The increase in the fluorescence signal over time for each collagenase standard concentration allowed the determination of the relationship between the gelatin digestion rate (RFU/min) and the presence of collagenase (ng/mL)

Fig. 5 Fig. 4
Fig. 5 Column chart plotting the activity of collagenases normalized by enzyme presence (ng/mL) quantified by Gel-FITC digestion in the 22 SF samples under study.Samples 6, 9 and 13 showed no collagenase activity signal.LLoD can be stablished at 8 ng/mL and ULoD can be stablished at 48 ng/mL

Table 1
Summary table of the most important types of MMPs in RA and OA, their names, and the types of collagens they digest

Table 2
The means, medians, maximum values, and minimum values of the presence of MMP-9 quantified by AbMAs in the 22 SF samples

Table 3
The mean, median, maximum value, and minimum value of the activity of collagenases normalized by enzyme presence (ng/mL) quantified by Gel-FITC digestion in the 22 SF samples

Table 4
Comparison between developed Gel-FITC collagenase activity assay and current techniques with the same intended use